Purification and properties of peritoneal eosinophils from pediatric dialysis patients.
Peritoneal eosinophilia frequently occurs in patients undergoing peritoneal dialysis. We have devised a method for isolating large numbers of these peritoneal eosinophils from pediatric patients on continuous peritoneal dialysis. Patients were selected on the basis of previous high peritoneal eosinophil counts and had an age range of 1.5-11 years. The unfractionated peritoneal fluid contained 7.9 +/- 3.7% neutrophils, 3.8 +/- 1.0% lymphocytes, 11.0 +/- 3.7% monocytes/macrophages, and 77.3 +/- 6.3% eosinophils (based on Wright stain) and up to 2 x 10(9) cells could be recovered from 1 liter of peritoneal dialysate. The cells were concentrated by centrifugation and the cell suspension then layered over a discontinuous Percoll gradient consisting of layers of 45%, 55%, 65%, and 75% Percoll. The gradients were centrifuged resulting in the formation of bands of cells at the interfaces of the layers. The densest band of cells (above 75% Percoll) contained 94.7 +/- 1.8% eosinophils (mean with median of 98%) and 4.3 +/- 16% neutrophils. The eosinophil counts were 72.2 +/- 7.1% above the 65% layer, 57.1 +/- 8.7 above 55%, and 40.9 +/- 10.9% above 45%. The monocyte/macrophage count increased from 0.1% above the 75% layer to 42.9% above the 45%. The denser eosinophils (above 75% and 65%) had the appearance of normal blood eosinophils and comparable function to blood eosinophils in cytotoxic and oxidative assays. This method provides a means of obtaining large numbers of very pure eosinophils for study of eosinophil function, eosinophil subpopulations, or eosinophil granule constituents.
Roberts
R L
RL
Department of Pediatric Immunology/Allergy, UCLA School of Medicine 90024.
Ank
B J
BJ
Salusky
I B
IB
Stiehm
E R
ER
D000954:Antigens, Surface / Q000276:immunology; D001965:Bromides / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002648:Child; D002675:Child, Preschool; D003601:Cytotoxicity Tests, Immunologic; D004802:Eosinophilia / Q000473:pathology*; D004804:Eosinophils / Q000276:immunology*; D005434:Flow Cytometry; D006098:Granulocytes / Q000276:immunology; D006801:Humans; D007223:Infant; D008163:Luminescent Measurements; D009504:Neutrophils / Q000276:immunology; D010529:Peritoneal Cavity / Q000473:pathology; D010531:Peritoneal Dialysis, Continuous Ambulatory
D000954:Antigens, Surface; D001965:Bromides